Std fimbriae-fucose interaction increases Salmonella-induced intestinal inflammation and prolongs colonization

RESEARCH ARTICLE Std fimbriae-fucose interaction increases Salmonella-induced intestinal inflammation and prolongs colonization Abdulhadi Suwandi 1☯ , Alibek GaleevID 1☯ , Rene ´RiedelID 2 , Samriti Sharma 1 , Katrin Seeger 1 , Torsten Sterzenbach 3 , Lucı ´a Garcı ´a Pastor 4 , Erin C. BoyleID 5,6 , Ohad Gal- MorID 7,8 , Michael HenselID 3 , Josep Casadesu ´s 4 , John F. Baines 2 , Guntram A. GrasslID 1 * 1Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School and German Center for Infection Research (DZIF), Hannover, Germany, 2Max Planck Institute for Evolutionary Biology, Evolutionary Genomics, Plo ¨n, Germany and Christian-Albrechts-University of Kiel, Kiel, Germany, 3Division of Microbiology and CellNanOs–Center for Cellular Nanoanalytics, University of Osnabru¨ck, Osnabru¨ck, Germany, 4Departamento de Gene ´tica, Facultad de Biologı ´a, Universidad de Sevilla, Sevilla, Spain, 5Institute for Laboratory Animal Science, Hannover Medical School, Hannover, Germany, 6Department of Cardiothoracic, Transplantation, and Vascular Surgery, Hannover Medical School, Hannover, Germany, 7The Infectious Diseases Research Laboratory, Sheba Medical Center, Tel-Hashomer, Israel, 8Department of Clinical Microbiology and Immunology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel ☯These authors contributed equally to this work. *[email protected] Abstract Expression of ABO and Lewis histo-blood group antigens by the gastrointestinal epithelium is governed by an α-1,2-fucosyltransferase enzyme encoded by the Fut2 gene. Alterations in mucin glycosylation have been associated with susceptibility to various bacterial and viral infections. Salmonella enterica serovar Typhimurium is a food-borne pathogen and a major cause of gastroenteritis. In order to determine the role of Fut2-dependent glycans in Salmonella-triggered intestinal inflammation, Fut2 +/+ and Fut2 -/- mice were orally infected with S. Typhimurium and bacterial colonization and intestinal inflammation were analyzed. Bacterial load in the intestine of Fut2 -/- mice was significantly lower compared to Fut2 +/+ mice. Analysis of histopathological changes revealed significantly lower levels of intestinal inflammation in Fut2 -/- mice compared to Fut2 +/+ mice and measurement of lipocalin-2 level in feces corroborated histopathological findings. Salmonella express fimbriae that assist in adherence of bacteria to host cells thereby facilitating their invasion. The std fimbrial operon of S. Typhimurium encodes the π-class Std fimbriae which bind terminal α(1,2)-fucose residues. An isogenic mutant of S. Typhimurium lacking Std fimbriae colonized Fut2 +/+ and Fut2 -/- mice to similar levels and resulted in similar intestinal inflammation. In vitro adhesion assays revealed that bacteria possessing Std fimbriae adhered significantly more to fucosylated cell lines or primary epithelial cells in comparison to cells lacking α(1,2)-fucose. Overall, these results indicate that Salmonella-triggered intestinal inflammation and colonization are dependent on Std-fucose interaction. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007915 July 22, 2019 1 / 22 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Suwandi A, Galeev A, Riedel R, Sharma S, Seeger K, Sterzenbach T, et al. (2019) Std fimbriaefucose interaction increases Salmonella-induced intestinal inflammation and prolongs colonization. PLoS Pathog 15(7): e1007915. https://doi.org/ 10.1371/journal.ppat.1007915 Editor: Andreas J. Baumler, University of California Davis School of Medicine, UNITED STATES Received: February 21, 2019 Accepted: June 13, 2019 Published: July 22, 2019 Copyright: ©2019 Suwandi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: This work was funded by the German Research Foundation (DFG) priority program SPP1656/1 and SPP1656/2 to GAG and JFB (http:// www.dfg.de). Work in the GAG lab is also supported by and German Federal Ministry of Education and Research (BMBF) Infect-ERA consortium grant 031L0093B (https://www.bmbf. de) and DFG collaborative research center SFB 900 Author summary The intestinal epithelium is a crucial biological interface, interacting with both commensal and pathogenic microorganisms. It’s lined with heavily glycosylated proteins and glycolipids which can act as both attachment sites and energy sources for intestinal bacteria. Fut2, the enzyme governing epithelial α1,2-fucosylation, has been implicated in the interaction between microbes and intestinal epithelial cells. Salmonella is one of the most important bacterial gastrointestinal pathogens affecting millions of people worldwide. Salmonella possesses fimbrial and non-fimbrial adhesins which can be used to adhere to host cells. Here we show that Salmonella expresses Std fimbriae in the gastrointestinal tract in vivo and exploit Std fimbriae to bind fucosylated structures in the mucus and on the intestinal epithelium. Furthermore, we demonstrate that the Std fimbriae-fucose interaction is necessary for bacterial colonization of the intestine and for triggering intestinal inflammation. These data lend new insights into bacterial adhesion-epithelial interactions which are essential for bacterial pathogenesis and key factors in determining tissue tropism and host susceptibility to infectious disease. Introduction Glycosylation is an important type of post-translational modification of proteins and lipids and is involved in the regulation of a wide range of processes at the cellular and molecular level. The gastrointestinal tract is home to a vast array of glycan structures and glycoconjugates [1], where the mucosal surface is the site of complex interactions between the intestinal microbiota, intestinal barrier, and immune system. The mucosal surface is characterized by a heavily glycosylated mucus layer produced by goblet cells as well as membrane-bound glycosylated proteins and lipids that form them [2]. These glycoconjugates can be utilized by intestinal commensal bacteria and pathogens as molecular attachment sites or as nutrients [3]. Importantly, host-derived glycans can help foster beneficial relationships with symbiotic microbes, such as Bacteroides thetaiotaomicron, by providing an energy source in the absence of dietary polysaccharides [4]. The FUT2 gene encodes the α-1,2-fucosyltransferase, a glycosyltransferase well known for its role in the expression of ABH and Lewis histo-blood group antigens on the gastrointestinal epithelium and in bodily secretions. Individuals expressing a functional allele are commonly described as ‘secretors’ whereas those homozygous for loss-of-function mutations display a ‘non-secretor’ phenotype. Variation in host glycosylation may directly influence susceptibility to enteric pathogens such as enterotoxigenic Escherichia coli [5], Helicobacter pylori [6], and norovirus [7]. Recent studies have shown the importance of host glycans in supporting a beneficial relationship with the endogenous microbiota by nourishing the microbiota during the stress of systemic infection [8] or by controlling opportunistic pathogens within the microbiota in the context of infection (e.g. Enterococcus faecalis) [9]. The non-secretor phenotype is also associated with an increased risk to develop chronic inflammatory bowel diseases [10]. This is possibly due to the altered composition of the intestinal microbiota, which may in turn influence the capacity of pathogenic bacteria to bind to host mucosal surface structures [11]. Salmonella enterica serovar Typhimurium (S. Typhimurium) is one of the most successful mucosal pathogens, colonizing the human gastrointestinal tract and causing severe inflammatory diarrhea [12]. S. Typhimurium carries several virulence genes including fimbrial adhesins, which are hair-like appendages on the outer membrane and are involved in adherence to host epithelial cells. Adhesion to host tissues is critical for invasion and pathogenicity of S. Salmonella fimbriae interaction with host glycans PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007915 July 22, 2019 2 / 22 TP8 (Projektnummer 158989968). Alibek Galeev and Samriti Sharma were supported by the Center for Infection Biology (ZIB) at the Hannover Biomedical Research School (HBRS). JFB was supported by DFG under Germany‘s Excellence Strategy – EXC 22167-390884018. MH was supported by DFG SFB 944, project P4 and BMBF Infect-ERA consortium grant 031L0093A. TS was funded by the DFG through grant TS 1921/3-1. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Typhimurium [13]. Type 1 fimbriae are one of the best characterized fimbrial adhesins and are encoded by the fim operon. FimH, a lectin-like protein, directly binds to high mannose oligosaccharides conjugated to surface glycoproteins of eukaryotic cells [14,15]. Another fimbrial operon, std, encodes the π-class Std fimbriae, which have been described to bind terminal α- 1,2 fucose residues [16]. The expression of bacterial adhesins possibly involved in binding fucosylated host proteins suggests that these fimbriae may facilitate Salmonella to establish or maintain infection in the highly fucosylated large intestine. Here, we investigated the role of host fucosylation in disease development during Salmonella infection using mice with and without expression of the Fut2 gene (Fut2 +/+ and Fut2 -/- ). Taken together, our results demonstrate that Std-fucose interaction contributes to S. Typhimurium persistence and inflammation. Results Fut2 expression affects susceptibility to Salmonella-induced colitis To test the hypothesis that expression of Fut2 influences host susceptibility to enteric pathogens, a model of S. Typhimurium-induced colitis was utilized. Fut2 +/+ and Fut2 -/- littermates were treated with streptomycin, and 24 hours later, infected with wild-type S. Typhimurium. One day post infection (p.i.), the cecal tissue of Fut2 -/- mice contained more S. Typhimurium than Fut2 +/+ mice (S1A Fig) in agreement with the observations of Goto and colleagues [17]. However, in contrast to their results, we found the total cecum weight and histopathology scores (S1B–S1D Fig) were comparable between Fut2 +/+ and Fut2 -/- mice. Under most conditions wild-type Salmonella kill C57BL/6 mice within approximately one week. Therefore, in order to follow the infection to later time points mice were infected with the S. Typhimurium ΔaroA mutant strain which is attenuated for systemic disease but causes extensive intestinal inflammation [18]. There was no significant difference in bacterial colonization or resulting inflammation of Fut2 +/+ and Fut2 -/- mice on day 1 p.i. (S1 Fig) or on day 3 p.i. (S2A–S2C Fig). However, on day 7 and day 14 p.i., a significantly reduced Salmonella burden in the intestine of Fut2 -/- compared to Fut2 +/+ mice was detected (Fig 1A,S2A–S2C Fig). Furthermore, 7 days p.i., the histopathological changes in the colon were significantly less severe in Fut2 -/- mice compared to Fut2 +/+ animals (Fig 1B and 1C). Notably, the colons of infected Fut2 +/+ mice were characterized by a higher number of detached epithelial cells within the colon lumen, increased inflammatory cell infiltration within the mucosa, and stronger submucosal edema. Additionally, the levels of the inflammation-associated marker lipocalin-2 were quantified in the large intestine after S. Typhimurium infection. The concentration of lipocalin-2 in the colon and cecum 7 days p.i. were significantly higher in Fut2 +/+ mice in comparison to Fut2 -/- (Fig 1D,S2D Fig). Next, colon tissue sections were analyzed by immunohistochemical staining and subsequent quantification of CD68- and MPO-positive cells, which represent macrophages and neutrophils, respectively. Consistent with elevated histopathological scores, significantly higher numbers of recruited neutrophils and macrophages were detected in the colon tissue of Fut2 +/+ mice compared to Fut2 -/- mice (Fig 2A–2C). In addition, a significantly stronger infiltration of CD4 + T lymphocytes in the colonic lamina propria of Fut2 +/+ mice compared to Fut2 -/- mice was detected by immunofluorescence staining and by flow cytometry (Fig 2A and 2D). No statistically significant differences were found in the numbers or infiltrate composition with respect to cytotoxic T lymphocytes, B lymphocytes, or dendritic cells by flow cytometric quantification of CD8 + , CD19 + , and CD11c hi cells, respectively (S3 Fig). To summarize, Fut2-expressing mice exhibited higher bacterial load in the intestine at later time points, which was also associated with an increase in inflammation assessed by Salmonella fimbriae interaction with host glycans PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007915 July 22, 2019 3 / 22 Fig 1. Fut2 expression affects susceptibility to Salmonella induced colitis. Streptomycin-treated mice were infected with S. Typhimurium ΔaroA for 7 days. (A) S. Typhimurium loads were determined in cecum tissue, cecum content, and colon tissue by plating homogenates on LB agar supplemented with streptomycin (n = 5 mice per group). (B) Histology scoring revealed higher inflammation in Fut2 +/+ -infected colons in comparison to Fut2 -/- -infected colons at day 7 post infection (p.i.). Uninfected colons of Fut2 +/+ and Fut2 -/- mice had low histology scores. (C) H&E staining of colon tissue sections at 7 days p.i. Scale bars, 50 μm. Fut2 +/+ and Fut2 -/- uninfected mice had a normal tissue and no signs of pathology. Higher numbers of cells in the lumen (L), an increased number of inflammatory cells in mucosa (M), elevated epithelial cell desquamation, and the formation of submucosal edema (E) upon S. Typhimurium infection were observed in Fut2 +/+ mice comparing to Fut2 -/- mice. (D) Lipocalin-2 levels measured by ELISA in supernatants of colon tissues homogenates (n = 5 per group) were higher in Fut2 +/+ mice compared to Fut2 -/- mice. Graphs are representative of three independent experiments. �p<0.05; ��p<0.01, Mann-Whitney test. https://doi.org/10.1371/journal.ppat.1007915.g001 Salmonella fimbriae interaction with host glycans PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007915 July 22, 2019 4 / 22 histopathology and lipocalin-2 levels, demonstrating that Fut2-mediated fucosylation in the intestine plays an important role in Salmonella-triggered inflammation and colonization of the intestine. Std fimbriae mediate adhesion to intestinal epithelial cells in a fucosedependent manner The Fut2 protein facilitates intestinal epithelial fucosylation by catalyzing the addition of L- fucose residues via an α(1,2) linkage to the terminal β-D-galactose residue of glycans. The std operon of S. Typhimurium encodes a fimbrial adhesin known to be important for the attachment to fucosylated structures on intestinal epithelial cells [16]. To investigate the role of fucose-Std fimbriae interaction during Salmonella adherence, human intestinal epithelial cell lines HT29-MTX-E12 and Caco-2 were utilized. HT29-MTX-E12 are colon epithelial cells that differentiate into goblet-like cells and produce mucus after three weeks of in vitro culture [19]. Ulex europaeus agglutinin I (UEA-I) lectin staining revealed extensive fucosylation of cell surface and mucus in the differentiated HT29-MTX-E12 cells in contrast to undifferentiated HT29-MTX-E12 cells. Wheat germ agglutinin (WGA) lectin staining for the ubiquitously expressed N-acetylglucosamine was positive in both differentiated and undifferentiated HT29-MTX-E12 cells (Fig 3A). Fig 2. Increased infiltration of immune cells in Fut2-expressing mice after Salmonella infection. (A) Immunofluorescence staining showed that Fut2 +/+ mice have higher numbers of CD3- (red) and CD68- (green) positive cells in the colon mucosa at day 7 p.i. Nuclei were stained with DAPI (blue). (B) Enumeration of stained cells indicated that Fut2 +/+ mice have higher numbers of CD68 + cells in colon mucosa at day 7 p.i. (C) MPO signal (red) showed increased recruitment of neutrophils in Fut2 +/+ mice. UEA-I (green) positive staining was detected only in Fut2 +/+ mice. Scale bars 50 μm. (D) Flow cytometry revealed a higher frequency of CD4 + (CD3 + CD4 + ) cells present in colonic lamina propria of Fut2-expressing mice compared to Fut2-deficient mice (n = 4–6). Scale bars, 50 μm. �p<0.05, Mann-Whitney test. https://doi.org/10.1371/journal.ppat.1007915.g002 Salmonella fimbriae interaction with host glycans PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007915 July 22, 2019 5 / 22 Previous studies have shown that bacterial expression of Std fimbriae is a subject of complex and tight regulation, both in vivo and in vitro [20,21]. Only a very small proportion of the Salmonella population express std fimbriae in vitro [22] and the std operon is completely absent in E.coli. Therefore, to analyze the role of Std fimbriae in vitro, an inducible expression plasmid containing the Salmonella stdABCD operon encoding the structural genes of Std fimbriae was transformed into a common laboratory E.coli K-12 and afimbriated E.coli ORN172. Upon induction with anhydrotetracycline, Std fimbriae were expressed by E.coli (referred as E.coli StdON) as confirmed by flow cytometry and Western blotting (Fig 3C and 3D). HT29-MTX-E12 cells were infected with either std-expressing (E.coli StdON) or non-express- ing bacteria (E.coli StdOFF). In contrast to E.coli StdOFF, only E.coli StdON strain showed increased adherence to differentiated HT29-MTX-E12 cells (Fig 3B). Importantly, expression of Std fimbriae had no effect on adhesion of E.coli StdON to undifferentiated HT29-MTX-E12 cells which do not contain fucosylated glycoproteins (Fig 3B). Similarly, E.coli ORN172 Fig 3. Std fimbriae mediate adhesion to intestinal epithelial cell culture in a fucose-dependent manner. (A) UEA-I (green) and WGA (red) staining in formalin-fixed HT29-MTX-E12 cells, at day 1 after seeding (undifferentiated) and at day 21 after seeding (differentiated). Positive UEA-I staining was present only in differentiated cells. Scale bars, 20 μm. (B) E.coli overexpressing stdABCD operon (StdON) displayed higher adherence to differentiated HT29-MTX-E12 cells; this effect was absent in differentiated HT29-MTX-E12 cells infected with E.coli StdOFF and in undifferentiated HT29-MTX-E12 cells infected with either strain. ���p<0.001; n.s. = not significant, ANOVA with Tukey’s multiple comparison test. (C) Flow cytometry analysis of std expression in E.coli strains. (D) Specificity of the serum and Std expression were additionally confirmed by Western blotting. E.coli RpoD (70kDa) was used as a control. https://doi.org/10.1371/journal.ppat.1007915.g003 Salmonella fimbriae interaction with host glycans PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007915 July 22, 2019 6 / 22 StdON bacteria adhered significantly better to Caco-2 cells compared to E.coli lacking Std expression. Addition of fucose-binding UEA-1 lectin to the cells prior to infection abrogated the adhesion of the std-expressing strain. In contrast, addition of dolichus biflorus aggluttinin (DBA), which binds to N-acetylgalactosamine, did not affect binding of E.coli ORN172 StdON to Caco-2 cells (S4A and S4B Fig). Atomic force microscopy showed Std piliation of E. coli ORN172 StdON and the absence of pili in the empty vector control bacteria (S4C Fig). In conclusion, Std fimbriae are important for binding α(1,2)-fucosylated residues on cell lines corroborating the results by Chessa and colleagues [16]. Next, Std-dependent bacterial adherence to primary epithelial cells was investigated. To this end, primary intestinal epithelial cells were isolated from Fut2 +/+ mice and cultivated as three-dimensional organoids in matrigel. These enteroids were expanded and seeded onto transwell filters resulting in the formation of a 2D monolayer consisting of various primary epithelial cell types. Monolayer barrier integrity and the degree of differentiation were evaluated by measuring transepithelial electrical resistance. Polarized monolayers were infected with E.coli StdON and StdOFF bacteria and adherence was analyzed by immunofluorescence. We counted the number of E.coli bacteria attached to UEA-1-positive and -negative cells. E. coli StdON bacteria were primarily associated with fucosylated cells, while E.coli StdOFF adhered equally to fucosylated and non-fucosylated cells (Fig 4A). Furthermore, a significantly higher number of the α(1,2)-fucose-associated E.coli StdON cells compared to the α(1,2)- fucose-associated E.coli StdOFF bacteria was detected (Fig 4B). Overall, this data demonstrate that std-expressing bacteria preferentially bind to fucosylated cells. Std fimbriae-fucose interaction is critical for Salmonella-induced inflammation and colonization Using cut sections of the cecum of CBA/J mice, it was previously demonstrated that purified Std fimbriae of S. Typhimurium are able to bind terminal α(1,2)-fucose residues in the mucosa [16]. However, the functional consequences of this interaction for disease development, as well as the extent of Std fimbriae expression in vivo are not known. To assess Std fimbriae production in vivo,stdA gene expression was first examined using RT-qPCR. Similar levels of stdA gene expression were detected in the colon of both Fut2 +/+ and Fut2 -/- mice infected with S. Typhimurium ΔaroA strain (S5A Fig). In order to determine whether the absence of std or the presence of intestinal fucosylated glycans affects expression of fucose or 1,2-propanediol utilization pathways we quantified levels of fucI and pduBC by RT-qPCR. We saw comparable levels of these genes expressed in either mouse strain in stdcontaining and std-deficient bacteria (S5B and S5C Fig). In order to look more closely at the spatial regulation of Std expression, we used reporter strains of S. Typhimurium containing a stdAstop::gfp fusion [22] and staining of tissue sections with anti-Std serum. Std was observed to be specifically expressed in the lumen of the large intestine of both Fut2 +/+ and Fut2 -/- mice on day 1 (S6 Fig) and day 7 p.i. (Fig 5 and S7 Fig). In contrast, Std-expressing Salmonella were not observed after invasion of the mucosa (Fig 5,S6 and S7 Figs). These data demonstrate that there is a tight spatial regulation of Std-expression whereby Std fimbriae are expressed prior to invasion of the large intestine. Fut2 -/- mice lack terminal fucose on intestinal epithelium [23]. To test whether epithelial fucosylation directly affects Salmonella colonization via interaction with Std fimbriae, the stdA mutation was transferred into the S. Typhimurium ΔaroA background strain. This mutant strain lacked functional Std fimbriae (S. Typhimurium ΔaroAΔstdA) yet had the same growth rate and motility as the parental S. Typhimurium ΔaroA strain (S8 Fig). Fut2 +/+ and Fut2 -/- mice were then infected with S. Typhimurium ΔaroAΔstdA via oral gavage for 7 days. In Salmonella fimbriae interaction with host glycans PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007915 July 22, 2019 7 / 22 contrast to S. Typhimurium ΔaroA (Fig 1), the S. Typhimurium ΔaroA ΔstdA strain colonized the colon of Fut2 +/+ and Fut2 -/- mice to similar levels (Fig 6A). In addition, histopathology scores showed similar intestinal inflammation of Fut2 +/+ and Fut2 -/- mice. H&E staining of colonic tissue showed moderate numbers of necrotic epithelial cells, mild inflammatory cell infiltration within intestinal mucosa and mild submucosal edema (Fig 6B and 6C), which was Fig 4. E.coli overexpressing Std fimbriae bind to fucosylated cells in primary intestinal epithelial monolayers. (A) 2D monolayers of Fut2 +/+ murine colon organoids were infected with either E.coli StdON or E.coli StdOFF and immunofluorescently stained using anti-E.coli and UEA-1 lectin. Image analysis of the infected monolayers revealed colocalization of Std-expressing E.coli bacteria (red) and the α(1,2)-fucosylated cells (green) (A, enlarged region and its orthogonal section). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. (B) Fucose-associated and non-associated E.coli were quantified microscopically using at least 20 fields of view (FOV) per sample. A significantly higher numbers of the fucoseassociated bacteria were detected when Std fimbriae were expressed. Data represents four independent biological repetitions. ���p<0.001; n.s. = not significant, ANOVA with Tukey’s multiple comparison test. https://doi.org/10.1371/journal.ppat.1007915.g004 Salmonella fimbriae interaction with host glycans PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007915 July 22, 2019 8 / 22 similar in both mouse genotypes. In addition, lipocalin-2 concentrations were similar in colons of both Fut2 +/+ and Fut2 -/- mice after infection with S. Typhimurium ΔaroAΔstdA (Fig 6D). Fig 5. Std fimbriae are differentially expressed in vivo.Fut2 +/+ (A) and Fut2 -/- (B) mice (n = 4) were infected with the reporter S. Typhimurium ΔaroA stdAstop::gfp strain. Mice were sacrificed at day 7 p.i. and colon sections were subjected to immunohistochemistry. GFP-positive bacteria were detected with an anti-GFP antibody. Std(GFP)-expressing S. Typhimurium were localized in the lumen of the colon, but not within the mucosa. Scale bars, 10 μm. https://doi.org/10.1371/journal.ppat.1007915.g005 Salmonella fimbriae interaction with host glycans PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007915 July 22, 2019 9 / 22 Immunofluorescence Formalin-fixed paraffin-embedded tissue sections (5 μm) were deparaffinized and rehydrated. Heat-induced epitope retrieval was performed using 10 mM sodium citrate buffer (pH 6.0) and blocking was achieved using 2% normal goat serum (NGS). The following antibodies were used for immunohistochemistry (see S1 Table for a full description): anti-StdA serum [38], Salmonella O Antiserum Group B (BD Difco), anti-GFP (DSHB), CD3 (Abcam), CD68 (Abcam), myeloperoxidase (MPO) (Thermo Fisher Scientific), and fluorescently-labeled secondary antibodies (Invitrogen). Counterstaining of nuclei was done with 4,6-Diamidin-2-phe- nylindol (DAPI) (Invitrogen). HT29-MTX-E12 and Caco-2 Bbe1 cells were seeded on coverslips in 24 well plates and fixed with 4% PFA before and after differentiation. Blocking of non-specific binding was done using 2% NGS. Fluorescently-labeled lectins UEA-1 (Ulex Europaeus agglutinin-1) (CosmoBio) and WGA (wheat germ agglutinin) (Vector laboratories) were used to visualize α(1,2)-fucosylation and the presence of sialic acid / N-acetylglucosami- nyl residues, respectively. Fixed primary epithelial cell monolayers were stained with the anti- E.coli antibody (Abcam), UEA-1 lectin (CosmoBio), and DAPI. Images were obtained on a Zeiss Apotome.2 microscope using AxioVision 4.9.1 software (Zeiss) and on a Leica DMi8 confocal laser scanning microscope using LAS X 3.3.0.16799 software (Leica). Brightness and contrast were adjusted using ImageJ 1.52e software. Flow cytometry Isolation of colonic lamina propria cells was achieved using the Lamina Propria Dissociation Kit (Miltenyi Biotec) according to the manufacturer’s protocol. Leukocyte isolation was performed with 40% / 80% discontinuous Percoll gradient (GE Healthcare). Cells were incubated with FcγR blocking reagent (rat anti-mouse CD16/CD32, BD Biosciences) for 30 minutes on ice prior to incubation with the other antibodies. Antibodies used for flow cytometry analysis are listed in S1 Table. To detect expression of Std fimbriae, flow cytometry was performed as previously described [16] with modifications. In brief, approximately 5x10 8 bacteria were fixed with 10% formalin and incubated at room temperature for 20 minutes. After washing with PBS, cells were resuspended in 2% NGS diluted in PBS and incubated at room temperature for 30 minutes. Polyclonal rabbit anti-StdA serum [38] was added to the cell suspensions following incubation at room temperature for 30 min. After washing with PBS, fluorescently labeled secondary antibodies (Invitrogen) were added. Flow cytometry was performed using a MACS- Quant Analyzer 10 (Miltenyi Biotec). The data were analyzed using FlowJo v.10 software (TreeStar). ELISA Supernatants from the organ homogenates were collected and stored at −20˚C. Lipocalin-2 levels were detected using mouse lipocalin-2/NGAL DuoSet ELISA (R&D Systems) according to the manufacturer’s protocol. Absorbance was measured using a Synergy HTX microplate reader and acquired using Gen5 software (Biotek). Western blot E.coli StdOFF and E.coli StdON were grown in LB broth supplemented with carbenicillin (50 μg/ml) and to induce std fimbrial expression anhydrotetracycline (100 ng/ml) at 37˚C until an OD 600 of 0.6 was reached. 10 8 bacteria were pelleted, resuspended in PBS, mixed with an equal volume of Laemmli buffer supplemented with 10% DTT, and boiled for 10 min. These whole-cell lysates were spun down and supernatants were loaded immediately onto a Salmonella fimbriae interaction with host glycans PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007915 July 22, 2019 16 / 22 SDS-PAGE gel (15%). Proteins were transferred to Hybond-P 0.45 PVDF (Amersham) membranes using a Trans-Blot semi-dry transfer cell (Bio-Rad). After blocking with Roti-block (Carl Roth), membranes were incubated first with anti-StdA [38] serum (Humphries et al., 2003) diluted 1:500 in blocking buffer, and then with a goat anti-rabbit-HRP conjugate, and finally with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific). Images were obtained using the ImageQuant LAS 4000 system (GE Healthcare). Salmonella gene expression in vivo Feces of the infected Fut2 +/+ and Fut2 -/- mice (day 7 p.i.) was immediately stored in RNAlater (Ambion). Total RNA was extracted using the High Pure RNA Tissue Kit (Roche) and reverse transcription was performed with the cDNA Synthesis Kit (Roche) in accordance with the manufacturer’s instructions. Quantitative real-time PCR (qPCR) was performed on a CFX96 Real-Time PCR Detection System (Bio-Rad) using the Power SYBR Green PCR Master Mix (Applied Biosystems). Gene-specific primers are listed in S2 Table. Data were normalized to the house-keeping gene rpoD and analyzed by ΔΔCt method [50] with median values of Fut2 +/+ mice infected with S. Tm ΔaroA as calibrators. Statistical analyses All data were analyzed using GraphPad Prism V7.0d software. Statistical analyses were performed using one-way analysis of variance followed by Tukey’s multiple comparison test or Wilcoxon-Mann-Whitney test as indicated. Graphs display the mean values ±SD, unless stated otherwise. Competitive index data were analyzed by the Wilcoxon signed-rank test by comparing medians with a hypothetical value of 1. Supporting information S1 Table. Antibodies and lectins used in this study. (DOCX) S2 Table. Primers used in this study. (DOCX) S1 Fig. Fut2-deficient mice are more susceptible to Salmonella infection on day 1 p.i. Streptomycin-treated mice were infected with WT S. Typhimurium SL1344 or S. Typhimurium ΔaroA and sacrificed at day 1 p.i. (A) S. Typhimurium loads were determined in the cecum by plating homogenates on LB agar with streptomycin (n = 4–5 mice per group). Higher bacterial loads were observed in Fut2 -/- mice infected with wildtype Salmonella. (B) Histology scoring revealed similar levels of inflammation in both Fut2 +/+ and Fut2 -/- ceca at day 1 p.i. (C) H&E staining of cecum tissue sections at 1 day p.i. Scale bars, 50 μm. Ceca of Fut2 +/+ and Fut2 -/- mice were characterized by high numbers of cells in the lumen (L), increased numbers of inflammatory cells in mucosa (M), massive epithelial cell desquamation, and the formation of submucosal edema (E) upon S. Typhimurium wildtype and ΔaroA infection. (D) Similar cecum weights in both Fut2 +/+ and Fut2 -/- mice were observed (n = 4–5 mice per group). �p<0.05; n.s = not significant, Mann-Whitney test. (TIF) S2 Fig. Fut2 +/+ mice carry higher bacterial burden at later infection time points. Streptomycin-treated Fut2 +/+ and Fut2 -/- mice were infected with S. Typhimurium ΔaroA for 3 and 14 days. (A-C) S. Typhimurium loads were determined in cecum tissue, cecum content and colon by plating homogenates on LB agar supplemented with streptomycin (n = 4–8 mice per Salmonella fimbriae interaction with host glycans PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007915 July 22, 2019 17 / 22 group). (D) Lipocalin-2 levels measured by ELISA in supernatants of cecum tissues homogenates (n = 5) were higher in Fut2 +/+ mice comparing to Fut2 -/- mice (day 7 p.i.). �P<0.05; n.s. = not significant, Mann-Whitney test. (TIF) S3 Fig. Immune cells in murine colon tissue after Salmonella infection at day 7 p.i. Similar frequencies of (A) CD19 + , (B) CD11c + and (C) CD3 + CD8 + cells were detected in colonic lamina propria of both Fut2 +/+ and Fut2 -/- using flow cytometry (n = 6 mice per group). n.s. = not significant, Mann-Whitney test. (TIF) S4 Fig. Std fimbriae mediate adhesion to intestinal epithelial cell culture in a fucose-depen- dent manner. (A) Caco-2 cells were infected with E.coli ORN172 expressing Std fimbriae (StdON) or not (empty vector). UEA-I (green), F-actin (purple) and StdA (red) staining of formalin-fixed Caco-2 cells displaying StdA-expression by StdON strain (top) and different degrees of fucosylation by Caco-2 cells. Scale bars, 20 μm. (B) E.coli ORN172 overexpressing Std (StdON) exhibited higher adherence to differentiated Caco-2 cells compared to E.coli ORN172 (empty). Adherence of E.coli StdON was abrogated upon addition of UEA-I lectin but not by addtion of DBA lectin prior to infection. ��p<0.002; n.s. = not significant, ANOVA with Tukey’s multiple comparison test. (C) Imaging of Std fimbriae on E.coli ORN172 StdON by atomic force microscopy (AFM). The height profile is indicated by heatmaps. Insert show 2.5-fold enlarged details of the cell envelope. (TIF) S5 Fig. Bacterial gene expression in vivo.Salmonella gene expression in feces of Fut2 +/+ and Fut2 -/- mice infected with either S. Typhimurium ΔaroA or S. Typhimurium ΔaroAΔstdA was measured. Gene expression was normalized to rpoD. Comparable levels of stdA expression was observed in both Fut2 +/+ and Fut2 -/- mice infected with S. Typhimurium ΔaroA (A). Neither fucI (B) nor pduBC (C) transcription was affected by Fut2 genotype or the presence or absence of stdAB genes. No significant differences were detected using one-way ANOVA with Tukey’s post test. (TIF) S6 Fig. Std fimbriae are differentially expressed in vivo on day 1 p.i. Fut2 +/+ and Fut2 -/- mice (n = 5 of each genotype) were infected with the reporter strain S. Typhimurium stdA- stop::gfp. Mice were sacrificed on day 1 p.i. and colon sections were stained with anti-GFP antibody to detect GFP-positive bacteria. Std(GFP)-expressing S. Typhimurium were found mostly in lumen of colon, and only few bacteria expressed Std within mucosa. Scale bars, 10 μm. (TIF) S7 Fig. Std producing Salmonella in vivo on day 7 p.i. stained with anti-Std antiserum. Colon sections of Fut2 +/+ and Fut2 -/- mice were stained with an anti-Salmonella antibody (green). A subset of Salmonella stained positive with anti-Std antiserum (red). Fucosylation was visualized with UEA-1 lectin staining (grey) and nuclei were stained with DAPI (blue). Scale bars, 10 μm. (TIF) S8 Fig. Deletion of stdAB genes does not affect motility and growth of S. Typhimurium. (A) S. Typhimurium SL1344 WT and S. Typhimurium SL1344 ΔstdA are similar in terms of motility. Deletion of stdAB genes has no effect on bacterial growth rate in WT (B) and ΔaroA Salmonella fimbriae interaction with host glycans PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007915 July 22, 2019 18 / 22 (C) backgrounds. (TIF) S9 Fig. Competitive index in feces. Competitive index (CI) was determined by infecting Fut2 +/+ and Fut2 -/- mice (n = 10 per group) with an equal amount of S. Typhimurium ΔaroA and S. Typhimurium ΔaroAΔstdA. Fecal homogenates from day 1, 3, 5 p.i. of both Fut2 +/+ and Fut2 -/- mice were plated on LB agar containing streptomycin (total Salmonella) and on LB plates with streptomycin+kanamycin (S. Typhimurium ΔaroAΔstdA only). Wilcoxon signedrank test, p values are indicated. (TIF) Acknowledgments We would like to thank Nicole Hansmeier for her support in atomic force microscopy imaging. Author Contributions Conceptualization: Abdulhadi Suwandi, Alibek Galeev, Rene ´Riedel, Torsten Sterzenbach, Lucı ´a Garcı ´a Pastor, Erin C. Boyle, Josep Casadesu ´s, John F. Baines, Guntram A. Grassl. Data curation: Abdulhadi Suwandi, Alibek Galeev, Rene ´Riedel, Samriti Sharma, Katrin Seeger, John F. Baines, Guntram A. Grassl. Formal analysis: Abdulhadi Suwandi, Alibek Galeev, Rene ´Riedel, Samriti Sharma, Katrin Seeger, Torsten Sterzenbach, Erin C. Boyle, Michael Hensel, John F. Baines, Guntram A. Grassl. Funding acquisition: Torsten Sterzenbach, Michael Hensel, John F. Baines, Guntram A. Grassl. Investigation: Abdulhadi Suwandi, Alibek Galeev, Katrin Seeger, Torsten Sterzenbach, Lucı ´a Garcı ´a Pastor, Erin C. Boyle, Michael Hensel, Josep Casadesu ´s, John F. Baines, Guntram A. Grassl. Methodology: Abdulhadi Suwandi, Alibek Galeev, Samriti Sharma, Katrin Seeger, Erin C. Boyle, Ohad Gal-Mor, Michael Hensel, Josep Casadesu ´s, John F. Baines, Guntram A. Grassl. Project administration: John F. Baines, Guntram A. Grassl. Resources: Torsten Sterzenbach, Lucı ´a Garcı ´a Pastor, Ohad Gal-Mor, Michael Hensel, Josep Casadesu ´s. Supervision: John F. Baines, Guntram A. Grassl. Validation: Abdulhadi Suwandi, Alibek Galeev, Rene ´Riedel, Samriti Sharma, Katrin Seeger, Torsten Sterzenbach, Lucı ´a Garcı ´a Pastor, Michael Hensel, Josep Casadesu ´s, John F. Baines, Guntram A. Grassl. Visualization: Abdulhadi Suwandi, Alibek Galeev, John F. Baines, Guntram A. Grassl. Writing – original draft: Abdulhadi Suwandi, Alibek Galeev, Rene ´Riedel, John F. Baines, Guntram A. Grassl. Salmonella fimbriae interaction with host glycans PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007915 July 22, 2019 19 / 22 Writing – review & editing: Abdulhadi Suwandi, Alibek Galeev, Rene ´Riedel, Samriti Sharma, Katrin Seeger, Torsten Sterzenbach, Lucı ´a Garcı ´a Pastor, Erin C. Boyle, Ohad Gal-Mor, Michael Hensel, Josep Casadesu ´s, John F. Baines, Guntram A. Grassl. 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